Targeted gene delivery to selected cell types provides a means for highly specific gene expression. Improved efficiency of gene transfer could be achieved through enhancing the entry of gene vectors into the desired cells and reducing the uptake of the vectors by non-target cells. For a therapeutic application, targeted gene delivery is critical in ensuring therapeutic effects in the cells of interest while limiting side effects, including immune, inflammatory and cytotoxic responses, caused by the expression of exogenous genes in non-target cells.
One approach for targeted gene delivery is to use ligand-associated delivery vectors. Via the ligands, the vectors recognize and bind to cell surface receptors that are unique to the target cells and that may undergo endocytosis upon binding to the ligands. The receptor-vector complexes, together with the surrounding plasma-membrane, may therefore become intracellular transport vesicles. Following gene escape from the vesicles and translocation of the gene to the nucleus, the gene product can be expressed. This receptor-mediated intracellular gene delivery was first reported by Wu G Y and Wu C H in 1987 (Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem 1987 Apr. 5; 262:4429–32; U.S. Pat. No. 5,166,320, granted Nov. 24, 1992) and has since received great attention in the field of drug/gene delivery. Many ligand-receptor systems have been investigated to date for targeted gene delivery. The ligands tested include asialoglycoproteins (Wu G Y and Wu C H, J Biol Chem., 262:4429–32, 1987), integrin-binding peptides (Berkner K L, Biotechniques 6: 616–629, 1988; Cristiano R J et al., Proc. Natl. Acad. Sci. USA, 90: 2122–2126,1993), transferrin (Zenke M et al., Proc. Natl. Acad. Sci. USA, 87: 3655–3659,1990; Wanger E, et al., Proc. Natl. Acad. Sci. USA, 88: 4255–4259,1991; U.S. Pat. No. 5,922,859), galactose (Remy J S et al., Proc. Natl. Acad. Sci. USA, 92:1744, 1995), folate (Lee R J and Huang L, J. Biol. Chem., 271, 8481, 1996), fibroblast growth factor (Goldman C K et al., Cancer Res, 57: 1447–1451, 1997; Hoganson D K et al., Human Gene Therapy, 9: 2565–2575, 1998), and epidermal growth factor (Schaffer and Lauffenburger, J Bio Chem, 273: 28004–28009, 1998). These ligands have been used to target mainly hepatocytes and tumor cells. The gene delivery systems based on the ligands are defined, in terms of their functions, by relatively well characterized ligand-receptor binding mechanisms involving related receptors. Very little has been established however for targeting gene vectors through receptor-ligand interaction to cells in a complex system, such as the nervous system.
Disorders in the nervous system, especially the central nervous system (CNS), such as stroke, epilepsy, head and spinal cord trauma, Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and many neurogenetic disorders, come with devastating effects on the individual and high social costs associated with chronic care and lost productivity. Many of the aforementioned disorders are related to the absence, malfunction or ineffectiveness of one gene or more and do not respond well to conventional therapeutic means. Gene transfer into the CNS has, therefore, been considered as a potential approach to treatment of these disorders. This approach may alter expression levels of neurotrophic factors, anti-apoptotic proteins, antioxidant molecules and other therapeutic factors to restore, halt or prevent the degeneration of cells in the CNS, especially neurons. Gene therapy also offers much hope for the treatment of CNS malignancies.
The unique characteristics of the CNS, the most sophisticated organ in the body, present several obstacles to successful gene therapy within the system. These characteristics include limited access to the CNS due to the physical barriers of the skull and the blood-brain barrier, the nature of terminally differentiated neurons and the difficulty of efficiently transfecting them with therapeutic genes. Moreover, the neuron types found within the CNS are very diverse, many of which are critical to physiological functions and highly sensitive to any kinds of changes. Under neurological situations, a disorder may affect one subtype of neurons and leave others unaffected. These features appeal strongly for the development of CNS gene therapy that restricts the expression of a therapeutic gene to a particular type of CNS cell, thus limiting possible side effects caused by gene expression in non-target CNS cells.
Currently, targeted gene delivery in the CNS is achieved simply through direct stereotactic injection of naked DNA or gene transfer vectors into well-defined anatomical locations, which may transduce various types of cells around the injection site. Retrograde axonal transport of viral or non-viral gene vectors offers another way to target neurons in different regions by choosing a proper injection site either in the CNS or the periphery. The method, however, transduce all projection neurons and does not distinguish subtypes of neurons.
Neurons are well known for their difference in the chemical transmitters that they use to deliver signals and in the receptors that they use to receive the signals. The nervous system makes use of 9 small-molecule transmitters and more than 50 neuroactive peptides, providing a way for classifying neurons based on their neurotransmitter phenotypes. In addition to their difference in sensitivities to various neurotransmitters, different neurons may also respond differently to another type of molecules called neurotrophic factors. These trophic factors are endogenous, soluble polypeptides displaying profound effects on the development, growth and survival of neurons and are believed to have great potential in the treatment of neurological disorders and CNS traumatic injuries. Among more than 20 known neurotrophic factors, the best-studied group is the neurotrophins, a family of structurally and functionally related polypeptides about 120 amino acids in length and sharing about 50% amino acid sequence identity. Four major neurotrophins isolated from mammals are nerve growth factor (NGF) (see SEQ ID NO:1 for human NGF sequence), brain-derived neurotrophic factor (BDNF) (see SEQ ID NO:2 for human BDNF sequence), neurotrophin 3 (NT3) (see SEQ ID NO:3 for human NT3 sequence) and neurotrophin 4/5 (NT4/5) (see SEQ ID NO:4 for human NT4/5 sequence). The neurotrophins are synthesized and secreted from neuronal target cells as non-covalently bound homodimers and act through retrograde signaling after binding to their nerve terminal-localized receptors. There are two main classes of receptors for the neurotrophins: the receptor protein tyrosine kinases including TrkA, TrkB and TrkC, and a 75-kDa transmembrane glycoprotein, p75NTR. NGF interacts selectively with TrkA, BDNF and NT4/5 primarily with TrkB and NT3 mainly with TrkC and, to a lesser extent, also with TrkA and TrkB (Meakin S O and Shooter E M, Trends Neurosci., 15: 323–331, 1992). All the neurotrophins interact with p75NTR, but with relatively low affinity as compared with their interaction with Trks.
Immunostaining reveals that Trks are expressed moderately in neurons and weakly in astrocytes in the adult human brain. No Trk staining is identified in oligodendrocytes. p75NTR is present in neurons, especially during development, in oligodendrocytes and at very low levels in many mature astrocytes. As a consequence of the Trk and p75NTR expression in various types of neurons, neurotrophins affect both overlapping and distinct subgroups of neurons (Thorne R G and Frey II W H, Clin Pharmacokinet, 40:907–946, 2001). Basal forebrain-cholinergic neurons respond to all four neurotrophins. Other neurons responsive to NGF include the striatal-cholinergic neurons, sympathetic sensory neurons, and neural crest-derived small-fibre sensory neurons. Other neurons responsive to BDNF include midbrain-dopaminergic neurons, spinal cord motor neurons, striatal-GABAergic and neural crest-derived medium-fibre sensory neurons and retinal ganglion cells. Other responsive neurons to NT3 include locus ceruleus neurons, midbrain-dopaminergic neurons, striatal-GABAergic neurons, sympathetic sensory neurons, and neural crest-derived large-fibre sensory neurons. The neurons responsive to NT4/5 also include locus ceruleus neurons, midbrain-dopaminergic neurons, striatal-GABAergic neurons, sympathetic and neural crest-derived sensory neurons, motor neurons and retinal ganglion cells. Outside the nervous system, NGF and TrkA expression is detected in B lymphocytes, T lymphocytes, mast cells, monocytes and macrophages, suggesting a role for NGF in immune and inflammatory functions. In addition, human tumor tissues up-regulate neurotrophin receptors and are responsive to NGF (Saragovi H U and Gehring K, TiPS, 21: 93–98, 2000).
Since the receptor-mediated endocytosis and retrograde transport of the ligand-receptor complexes are essential to neurotrophin signaling within the aforementioned cells (Neet K E and Campenot R B, CMLS Cell. Mol. Life Sci., 58:1021–1035, 2001), these polypeptides are candidates for targeting ligands that can be used to target gene vectors to Trk or p75NTR positive cells. For example, native neurotrophin polypeptides can be chemically conjugated to a cationic lipid or polymer that has the capability to bind to and condense DNA. This procedure, however, requires relatively larger amounts of purified polypeptides and may involve harsh chemical reactions that may significantly reduce bioactivities of the polypeptides. Recombinant DNA technology is one of the common methods used to produce biologically active, full-length neurotrophin polypeptides, as described in U.S. Pat. No. 5,606,031 and U.S. Pat. No. 6,005,081. Using these techniques in a bacterial expression system, misfolded variants of neurotrophins, differing from their authentic forms by the incorrect pairing of their cysteine residues, constitute a major problem. Neurotrophin polypeptides have similar structures that contain cystine knot motifs, with six residues of cysteine at highly conserved positions. The formation of correctly paired intramolecular disulfide bonds is required for full biological activity of a neurotrophin. A study using E. Coli to express BDNF has reported ten protein variants with incorrectly-paired disulfide linkages among six residues of cysteine, and all of them had much lower biological activity than that of native BDNF and may significantly inhibit the biological activity of authentic BDNF when used together (Shimizu N et al., Biosci. Biotech. Biochem., 60: 971–974, 1996). Other potential problems preventing the efficient use of a large protein as a targeting ligand include proteolytic degradation tendency, immunogenecity, and poor pharmacokinetics.
While all neurotrophins share almost the same core structure composed of a pair of two-stranded, twisted β-sheets, there are seven distinct regions in the proteins, including N terminus, loop regions I, II, III, β strand region IV, loop region V and C terminus, with higher than average sequence diversity (Ibanez C F, TIBTECH, 13: 217–227, 1995). Various approaches, from site-directed mutagenesis, deletion, chimeric molecule construction to crystal structure analysis, have been used to study structural determinants of neurotrophin effects. These studies have confirmed the amino acid residues that are important for binding to Trk receptors are localized, in the case of NGF, in N-terminal region (#1–8), loop region II (#40–49) and loop region V (#96–97) (Ibanez C F, TIBTECH, 13: 217–227, 1995). The study on the crystal structure of NGF in complex with the immunoglobulin-like domain 5 of TrkA (Wiesmann C et al., Nature, 401: 184–188, 1999) brought to attention on two patches; one patch involving the core beta-sheet of the homodimeric NGF molecule and another patch comprising the N-terminal residues of NGF for specific interaction with TrkA. Small peptide mimetics of NGF have been reported to activate TrkA-related signal transduction and promote NGF-like neurotrophic effects (U.S. Pat. No. 5,958,875; U.S. Pat. No. 6,017,878; Beglove N et al., J Med Chem., 43: 3530–3540, 2000; Maliartchouk S et al., J. Bio. Chem., 275: 9946–9956, 2000; Xie Y et al., J Bio. Chem, 275: 29868–29874, 2000). These peptide mimetics need to be cyclic, not only being sequence analogs but also structure analogs of NGF loops, in order to be biologically active.